The broad objective of this project is to understand how RNA and protein molecules assemble to form native ribonucleoprotein complexes and collaborate to perform RNA-based catalysis. In the first system, we will explore assembly of the bl5 ribonucleoprotein, consisting of the bl5 group I intron RNA and its single obligatory protein cofactor, CBP2. In particular, we will use this system to understand the structure of the RNA collapsed state, identified in the P.l.'s lab, that forms prior to subsequent assembly with the CBP2 protein cofactor. It is becoming clear that related collapsed states play fundamental, productive, roles in many RNA folding and ribonucleoprotein assembly reactions. The second system is the bl3 group I intron ribonucleoprotein. Splicing by the bl3 intron requires two facilitating proteins. The intron-encoded bl3 maturase binds a monomer; whereas, two Mrs1 dimers bind, cooperatively, to yield a six-component complex. The bl3 ribonucleoprotein will be developed as a model for understanding ribonucleoproteins that require binding by multiple protein cofactors to achieve a biologically functional state. Both simple systems are sufficiently sophisticated to illustrate principles generalizable to biomedically important ribonucleoproteins like the ribosome and pre-mRNA complexes. Specific aims are: (1) To determine the physical basis by which the collapsed state functions to prevent mis-assembly of the bl5 ribonucleoprotein. (2) To study the structure of the bl5 RNA under conditions likely to predominate in yeast mitochondria. (3) To understand the molecular basis for the predominance of near-native structures in the bl5 collapsed state. (4) To develop a comprehensive kinetic and thermodynamic framework for assembly of the six-component bl3 group I intron RNP catalyst. (5) To begin to explore the molecular mechanism by which Mrs1 facilitates catalysis of the bl3 group I intron. (6) To develop a high resolution model for the maturase-bl3 RNA complex.